The present invention relates to purified hCG-hLH receptor, hCG-hLH receptor-hCG complex and combinations between their subunits as antigens, as well as antibodies thereto which are useful as a contraceptive vaccine and nucleotide sequences encoding polypeptides with receptor activity.
In recent years significant effort has been expended toward developing an immunological approach to contraception. The basic approach has been to either provide an antibody (passive immunization), or to elicit an antibody response (active immunization), to a hormone critical to the establishment and/or maintenance of pregnancy. The production and effects of human chorionic gonadotropin (hCG) in pregnancy have singled out hCG as a prime candidate for studies in immunological contraception. hCG is not present in the normal, healthy female prior to fertilization, but is secreted by the developing blastocyst and can be detected in pregnant women as early as 6 to 7 days after fertilization. hCG, in turn, initially acts upon the corpus luteum, and later upon the placenta, in causing each of them to secrete progesterone. Progesterone, at a high level, acts upon the endometrium to aid in preparing it for implantation and to maintain it after implantation. Therefore, both hCG and progesterone are essential for pregnancy to proceed immediately following fertilization. However, a significant reduction of hCG level prevents sufficient hCG from interacting with the hCG receptors of the corpus luteum and the placenta for maintenance of the high level of progesterone required for pregnancy. Progesterone drops back to or remains at a level too low for support of the endometrium, in the absence of hCG.
A number of researchers have attempted to develop contraceptive vaccines which immunologically block progesterone production. These vaccines provide or produce hCG antibodies to immunologically interact with circulating hCG determinants, thereby preventing the hCG determinants from reaching the hCG receptors of the corpus luteum and of the placenta.
Various problems have prevented commercialization of an hCG vaccine. First, hCG is a human hormone and humans will not normally produce antibody to a human hormone. This problem has been attacked by linking the hCG to a protein such as a hapten. Of course, the hCG antibodies can be produced in normal fashion in animals such as rabbits. However, problems still occur due to the non-specificity of hCG antibody, i.e., high levels of hCG antibody cross react with human luteinizing hormone (hLH); high levels of hCG antibody tend to cause abortion; etc. Low levels of hCG antibodies, which would not cross react with hLH, i.e., are hCG-xcex2 specific, were thought to offer the best chance of success, but in practice the circulating life of hCG is extended by formation of loose antigen-antibody complexes.
hCG and hLH, which share common receptors in the gonads as well as follicle stimulatory hormone (FSH), play important roles in the growth of ovarian follicles and in spermatogenesis in the testes.
A group of patents by Bahl (U.S. Pat. No. 4,310,455 and others) concern modification of the xcex2 subunit of hCG to produce a more specific hCG antigen for a variety of uses, including a contraceptive vaccine.
U.S. Pat. No. 4,161,519 by Talwar discloses a contraceptive vaccine comprising a purified xcex2 subunit of hCG conjugated to an antigen carrier.
A number of papers related to the general subject of contraception based on tying-up circulating hCG are found in Recent Advances in Reproduction and Regulation of Fertility, Elsevier/North Holland, 1977 (G. P. Talwar, Editor), pages 427-485.
Luborsky and Behrman (Biochem. Biophys. Res. Comm. 90, 1407, 1979) used rat ovary detergent lysates as a source of LH-R. Polyclonal antibodies thereto reacted with rat gonadal tissues but not with human or sheep ovary. The antiserum blocked LH-induced progesterone secretion by rat luteal cells.
Metsikko and Rajaniemi (Endocrinology 109, 1399, 1981; Biochem. J. 224, 467, 1984) prepared an antiserum to purified LH receptor from rat ovarian tissue. The receptor was presaturated with hCG prior to use as immunogen. The resulting antiserum immunoprecipitated a polypeptide of about 95,000 molecular weight. Those findings are at odds with that reported in the instant invention.
Podesta et al. (Proc. Natl. Acad. Sci. 80, 3986, 1983) described a monoclonal antibody raised to a rat ovarian membrane preparation. The antibodies inhibited testosterone production by isolated Leydig cells exposed to LH.
Rosemblit et al. (Endocrinology 123, 2284, 1988) described a polyclonal antiserum raised to purified rat LH receptor. The receptor had a molecular weight of about 93,000 daltons. Rosemblit et al. distinguished their antibody over that of Metsikko and Rajaniemi and of Podesta et al.
Vuhai-Luuthi et al. (Endocrinology 127, 2090, 1990) described monoclonal antibodies directed to porcine LH receptor complexed with human chorionic gonadotrophin. The antibody immunoprecipitated a major protein of 85,000 molecular weight, and two minor proteins of approximately 68,000 molecular weight and about 45,000 molecular weight. The 85,000 molecular weight protein was found in both testicular membrane extracts and ovarian membrane extracts.
Therefore, it is an object of the present invention to provide an improved contraceptive vaccine.
A more specific object of this invention is to provide a contraceptive vaccine which functions by preventing hCG from stimulating progesterone production of the corpus luteum and/or placenta.
Still another object of the present invention is to provide a contraceptive vaccine which overcomes the problems encountered with only hCG-based vaccines of the prior art.
A further object of the present invention is to provide contraceptive vaccines which can be used for either passive or active immunization.
Another object of the invention is to provide antibodies to LH-R which can modulate sex hormone secretion.
Yet another object of the instant invention is to provide nucleotide sequences that encode polypeptides capable of binding hCG and hLH and bindable by xcex1-receptor antibodies.
Since hCG and hLH have common receptors an additional object of the present prevention is to use hCG-hLH receptor as an antigen and antibodies thereto in order to reversibly retard ovarian follicular growth and corpus luteum function which is believed to prevent ovulation and thus fertility.
Other objects of the invention, such as the provision of novel antigens and antibodies and methods to obtain such, will be apparent to the skilled artisan from the Detailed Description of the Invention, hereinbelow.
In accordance with the present invention, there is provided a contraceptive vaccine based on hCG, or a derivative; fragment or subunit thereof, and the common receptor for hLH and hCG, or a derivative, fragment or subunit thereof. In preferred embodiments of the invention, a dual purpose antigen is formed by complexing or conjugating the hCG-xcex2 subunit, or a derivative or fragment thereof, to the common biological receptor for hCG and hLH, or a derivative, fragment or subunit thereof. In active immunization embodiments, the two antigen components used in the present invention are administered either separately or in the form of the above-described complex or conjugate. In passive immunization embodiments, the antigen materials, separately or as the conjugate, are administered to a lower animal for production of antibodies, which are collected in the usual fashion and administered as a vaccine. In the most preferred embodiments of the present invention, only the common receptor for hLH and hCG, or a derivative, fragment or subunit thereof, is employed for either active or passive immunization as well as for the production of monoclonal antibodies for use as contraceptive agents.
As noted above, in the preferred embodiments of the invention, hCG-xcex2 is linked or complexed with the biological receptor for hCG and hLH (hereinafter xe2x80x9creceptorxe2x80x9d) to form an antigen hCG-receptor unit capable of circulation in the bloodstream as an integral moiety. As disclosed herein, either the antigen hCG-receptor unit can be administered as a contraceptive vaccine as in the preferred embodiment of the invention or the antigen receptor only can be administered as a contraceptive vaccine as in the most preferred embodiment of the present invention, or in another embodiment at this time, antibody to either the hCG-receptor antigen or the receptor antigen can be administered as the contraceptive vaccine.
The common receptor for hLH and hCG is antigenic, so that the antibodies produced in response to the hCG-receptor unit contain determinants for both hCG and the receptor. In this manner, some of the antibodies of this invention not only interact with hCG to prevent hCG from reaching the receptors of the corpus luteum and placenta, but also blocks the receptor sites, thereby preventing any remaining unbound hCG from reaching the receptor sites.
In preferred embodiments of the invention, the hCG antigen consists essentially of the hCG-xcex2 subunit, or a derivative or fragment thereof, intact or modified.
In other preferred embodiments of the invention, the antibodies of the invention contain essentially monospecific determinants for the hCG-xcex2 subunit and essentially monospecific determinants for the receptor.
In passive immunization of the present invention, bifunctional and/or mono-functional polyclonal or monoclonal antibodies may be involved as well as idiotypic antibodies.